Volume 4, Issue 6, Pages 603-613 (June 1996) Normal Human IgD+IgM− Germinal Center B Cells Can Express Up to 80 Mutations in the Variable Region of Their IgD Transcripts  Yong-Jun Liu, Odette de Bouteiller, Christophe Arpin, Francine Brière, Laurent Galibert, Stephen Ho, Hector Martinez-Valdez, Jacques Banchereau, Serge Lebecque  Immunity  Volume 4, Issue 6, Pages 603-613 (June 1996) DOI: 10.1016/S1074-7613(00)80486-0

Figure 1 Identification of sIgD+CD38+ Tonsillar B Cells (A) Double staining of total tonsil B cells with anti-IgD–PE and anti-CD38–FITC, showing four B cell subsets. The sIgD+CD38+ B cells represent 5%–15% of total tonsil B cells from more than 30 samples analyzed to date. (B) Phenotypic analysis by three-color flow cytometry. Anti-IgD–tricolor and anti-CD38–PE are used together with other third antibodies directly conjugated with FITC. Anti-IgD–PE and anti-CD38–FITC were used together with Hoechst 33342. For Bcl-2 and Ki67 staining, the cells were permeabilized by saponin (0.33 g/100 ml in PBS 1% BSA) for 15 min on ice. Immunity 1996 4, 603-613DOI: (10.1016/S1074-7613(00)80486-0)

Figure 2 PCR Products of VH5-μ, VH5-δ, and VH5-γ on Ethidium Bromide–Stained Agarose Gel sIgD+CD38− naive B cells and sIgD+CD38+ B cells only express μ and δ transcripts, while sIgD−CD38− GC B cells only express μ and γ transcripts. Immunity 1996 4, 603-613DOI: (10.1016/S1074-7613(00)80486-0)

Figure 3 VH5-δ Sequences from sIgD+CD38− Naive B Cells, sIgD+CD38+ GC B cells, and the VH5-γ Sequences from sIgD−CD38+ GC B Cells The upper and lower groups of sequences represent two different tonsils. Mutations are represented by replacement mutation (closed circle with stem), silent mutation (stem), and hot spot of somatic mutation (open circle with stem). Each line represents one VH5 sequence. VH5-2 sequences are underlined. Sequences 4b and 4b′ derive from two independent reverse transcriptions and PCRs of the same RNA sample. Clonally related sequences are boxed. Mutations present in the DJH regions are not shown. 3′ Cδ regions are unmutated. Immunity 1996 4, 603-613DOI: (10.1016/S1074-7613(00)80486-0)

Figure 4 VH5-δ Sequences from sIgM−sIgD+CD38+ GC B Cells and from sIgM−sIgD+ Peripheral Blood B Cells sIgD+CD38+ B cells from a third tonsil were separated into sIgM+ and sIgM− subsets. The VH5-δ sequences from the sIgM+ subset are either in germline or low mutated (data not shown). The VH5-δ sequences from the sIgM− subset are shown in the right panel. sIgM−IgD+ B cells were isolated from two peripheral blood samples. Their VH5-δ sequences are shown in the left panel. The method for sequencing is detailed in Pascual et al. 1994. Immunity 1996 4, 603-613DOI: (10.1016/S1074-7613(00)80486-0)

Figure 5 Sequence Analysis of the Two Largest Hypermutated Clones (A and B) Nucleotide sequences of hypermutated VH5-δ clone 4b.1 and clone 6c.1. (C) Genealogical trees from clone 4b.1 and clone 6c.1. The germline VH5–D–JH sequence (GL) was deduced from the consensus sequence for each clone. Clonal trees were drawn so as to minimize the number of parallel mutations. Each analyzed sequence is indicated by its circled number, while common predicted intermediates are boxed. The length of each line is proportional to the number of mutations (indicated in the figure) that separate two connected sequences. Immunity 1996 4, 603-613DOI: (10.1016/S1074-7613(00)80486-0)

Figure 5 Sequence Analysis of the Two Largest Hypermutated Clones (A and B) Nucleotide sequences of hypermutated VH5-δ clone 4b.1 and clone 6c.1. (C) Genealogical trees from clone 4b.1 and clone 6c.1. The germline VH5–D–JH sequence (GL) was deduced from the consensus sequence for each clone. Clonal trees were drawn so as to minimize the number of parallel mutations. Each analyzed sequence is indicated by its circled number, while common predicted intermediates are boxed. The length of each line is proportional to the number of mutations (indicated in the figure) that separate two connected sequences. Immunity 1996 4, 603-613DOI: (10.1016/S1074-7613(00)80486-0)

Figure 5 Sequence Analysis of the Two Largest Hypermutated Clones (A and B) Nucleotide sequences of hypermutated VH5-δ clone 4b.1 and clone 6c.1. (C) Genealogical trees from clone 4b.1 and clone 6c.1. The germline VH5–D–JH sequence (GL) was deduced from the consensus sequence for each clone. Clonal trees were drawn so as to minimize the number of parallel mutations. Each analyzed sequence is indicated by its circled number, while common predicted intermediates are boxed. The length of each line is proportional to the number of mutations (indicated in the figure) that separate two connected sequences. Immunity 1996 4, 603-613DOI: (10.1016/S1074-7613(00)80486-0)

Figure 6 Morphological Characterization of B Cell Subsets (a) Giemsa staining of freshly isolated IgD+CD38− small dense naive B cells. (b) Giemsa staining of freshly isolated IgM−IgD+CD38+ large cells, displaying pyroninophilic scanty cytoplasm and blastic nuclear with multiple nucleoli, typical of centroblast morphology. (c) Red anti-Ki67 staining showing sIgM−IgD+CD38+ GC B cells expressing a high level of Ki67 antigen, a centroblast marker. (d) Giemsa staining of sIgM−IgD+CD38+ GC B cells after 16 hr culture, displaying apoptotic figures. Hypermutated sIgM−IgD+CD38+ GC cells (5 × 105) were cultured for 5 days with (e) CD40 ligand-transfected L cells; (f) CD40 ligand-transfected L cells, human fibroblasts from rheumatoid synovium, IL-10, and culture supernatant from human CD4+ T cells activated with anti-CD2, anti-CD28, and PMA for 8 hr. (Culture conditions are detailed in Figure 7.) Immunity 1996 4, 603-613DOI: (10.1016/S1074-7613(00)80486-0)

Figure 7 DNA Synthesis by sIgM+IgD+ CD38+ and sIgM−IgD+CD38+ GC B Cells Cells (2.5 × 105/ml) were cultured for 5 days in different conditions. IL-2 (10 U/ml), IL-4 (50 U/ml), IL-10 (100 ng/ml), 50% supernatant from human peripheral blood CD4+ T cells activated for 8 hr with anti-CD2, anti-CD28, and PMA, 2.5 × 104 CD40 ligand-transfected L cells, and 2.5 × 103 human fibroblasts from rheumatoid synovium (irradiated with 75 Gy) were used for the cultures. DNA synthesis was assessed by a 8 hr pulse with 1 μCi [3H]TdR before cell harvesting. Immunity 1996 4, 603-613DOI: (10.1016/S1074-7613(00)80486-0)

Figure 8 Localization of IgM−IgD+CD38+ GC B Cells in Tonsil Sections (a) Red anti-IgD staining showing a red IgD+ mantle zone (FM) and a GC of a secondary follicle. Some IgD+ B cells are identified within the GC, mainly in the dark zone. Original magnification, 100×. (b) Double anti-IgD (red) and anti-CD38 (blue) staining on an adjacent section, showing IgD+CD38+ B cells in the GC dark zone shown in (a). Original magnification, 200×. (c) Double anti-IgD (red) and anti-Ki67 (blue) staining of the same GC dark zone (original magnification, 200×). Nearly all IgD+ B cells within the GC dark zone are Ki67+, in contrast with the IgD+ B cells in the mantle zone (data not shown) and extrafollicular areas. (d) A GC does not contain IgD+ B cells. Original magnification, 100×. (e) Red anti-IgD and blue anti-IgM staining showing small purple IgM+IgD+ follicular mantle B cells; blue IgM immune complexes within the GC light zone; and many large red IgM−IgD+ B cells in the GC dark zone. Original magnification, 100×. (f) The GC dark zone shown in (e) at an original magnification of 200×, showing small purple IgM+IgD+ B cells and large red IgM−IgD+ B cells. Immunity 1996 4, 603-613DOI: (10.1016/S1074-7613(00)80486-0)